Targeted genome modification of plants has been a long-standing and elusive goal of both applied and basic research. In principle, the ability to direct transgene integration to specific sites within the plant genome and to make precise nucleotide sequence alterations would not only provide a powerful tool for basic studies of plant gene function but would also directly enhance the development of new crop varieties. However, current approaches to plant genome modification involve either the random integration of DNA into arbitrary genomic locations, as originally described in 1983, or the indiscriminant alteration of gene sequences with chemical or physical mutagens.
Although well established in yeast and moss, gene targeting—the introduction of foreign DNA into a predetermined genomic location—remains a significant challenge in higher plants. Site-specific transgene integration occurs at a very low frequency in plant cells as compared to random integration, even when the incoming DNA contains large stretches of sequence homologous to host DNA. For example, a highly efficient Agrobacterium-based transfection system and herbicide selection resulted in gene targeting frequencies of up to 5×10−4 in rice. Attempts to enhance gene targeting efficiencies in plants have included the use of negative selection markers, and the use of plants genetically engineered to exhibit higher targeting frequencies. These efforts notwithstanding, random DNA integration via non-homologous processes continues to be a major impediment to gene targeting in plants. Given the general utility envisioned for targeted gene addition in the modification of crops for agricultural and industrial biotechnology, a solution to this problem is sorely needed.
In this regard, substantial increases in the frequency of gene targeting in a broad range of plant and animal model systems have been observed following the induction of a DNA double-strand break (DSB) at a specific genomic location in host cells, which stimulates a native cellular process, homology-directed DSB repair. Naturally occurring site-specific endonucleases whose recognition sites are rare in the plant genome have been used in this manner to drive transgene integration into a target sequence previously transferred into the plant genome via random integration. These studies highlighted the potential of targeted DSB induction to stimulate gene targeting in plant cells, though the challenge of introducing a DSB in a native locus remains.
In animal cells, the solution to targeted induction of a DSB at a native genomic location is provided by zinc finger nucleases (ZFNs). The C2H2 zinc finger was discovered in the amphibian transcription factor TFIIIA, and has since been found to be the most common DNA recognition motif in all species of metazoa. The X-ray crystal structure of the C2H2 ZFP, Zif268, revealed a strikingly syllabic mode of protein-DNA recognition, with each zinc finger specifying a 3 or 4 bp subsite in the context of a tandem arrangement, and suggested the possibility of using this peptide motif as a scaffold for DNA binding domains with novel specificities. Since then, a large number of ZFPs engineered to bind novel sequences have been successfully used in many different laboratories in the context of artificial transcription factors and other functional chimeric proteins.
Zinc finger nucleases are produced by fusing a zinc finger protein with a sequence-independent nuclease domain derived from the Type IIS restriction endonuclease FokI. Beginning with studies in Xenopus and fruit flies, a DSB targeted by ZFNs to an investigator-specified DNA sequence has been shown to stimulate homology-directed DNA repair in a range of model systems. More recently, engineered zinc finger nucleases have emerged as flexible and effective tools for native gene correction and disruption in human, hamster, nematode, and zebrafish. Moreover, and of relevance to the current work, ZFNs have been used to drive high-efficiency targeting (or “gene addition”) to a native locus without any measurable increase in the rate of random integration, initially in transfected and subsequently in primary human cells.
Importantly, initial attempts at using ZFNs in plants have been equally successful. In Arabidopsis, ZFNs have been demonstrated to introduce targeted mutations at frequencies as high as 20%. Furthermore, in tobacco, using a pre-engineered target site, it was shown that zinc finger nucleases may target specific sites pre-integrated into a plant genome and facilitate site-specific DNA integration, in agreement with findings made with endonucleases such as I-SceI.